Quantitative single-molecule FLIM and PIE-FRET imaging of biomolecular systems

The structural dynamics of proteins and nucleic acids are critical for their function in many biological processes, but investigating these dynamics is often challenging with traditional techniques. Time-correlated single photon counting (TCSPC) coupled with confocal microscopy is a versatile biophysical tool that enables real-time monitoring of biomolecular dynamics in a variety of systems, across many timescales. Quantitative single-molecule time-resolved fluorescence methods are uniquely positioned to investigate transient interactions and structural changes, yet application in complex biological systems remains limited by technical and analytical challenges. Combining fluorescence lifetime imaging microscopy (FLIM) with pulsed interleaved excitation Förster resonance energy transfer (PIE-FRET) offers a robust approach to overcome these barriers, enabling accurate distance measurements and dynamic studies across diverse sample types. In this study, we described practical workflows fo..., , # Data from: Quantitative single-molecule FLIM and PIE-FRET imaging of biomolecular systems

Dataset DOI: [10.5061/dryad.zkh1893pv](https://doi.org/10.5061/dryad.zkh1893pv)

## Description of the data and file structure

We describe practical workflows for implementing FLIM/PIE-FRET for quantitative measurements of nanoscale distances and dynamic processes in various biomolecular systems on a commercial microscope.

### Files and variables

#### File: Hi_10pM_17_0712_acc.dat

**Description:** High FRET benchmark DNA single-molecule acceptor trace 

#### File: Hi_10pM_17_0712_donor.dat

**Description:** High FRET benchmark DNA single-molecule donor trace 

#### File: Lo_10pM_23_0821_DO.dat

**Description:** Lo FRET benchmark DNA single-molecule donor only trace 

#### File: Hi_10pM_27_0712_DO.dat

**Description:** Hi FRET benchmark DNA single molecule donor only trace 

#### File: Lo_10pM_27_0712_acc.dat

**Description:** Lo FRET benchmark DNA single-molecule acceptor trace 

#### File: ...,